Preparation and Biological Study of 68Ga-DOTA-alendronate

Objective(s): In line with previous research on the development of conjugated bisphosphonate ligands as new bone-avid agents, in this study, DOTA-conjugated alendronate (DOTA-ALN) was synthesized and evaluated after labeling with gallium-68 (68Ga). Methods: DOTA-ALN was synthesized and characterized, followed by 68Ga-DOTA-ALN preparation, using DOTA-ALN and 68GaCl3 (pH: 4-5) at 92-95° C for 10 min. Stability tests, hydroxyapatite assay, partition coefficient calculation, biodistribution studies, and imaging were performed on the developed agent in normal rats. Results: The complex was prepared with high radiochemical purity (>99% as depicted by radio thin-layer chromatography; specific activity: 310-320 GBq/mmol) after solid phase purification and was stabilized for up to 90 min with a log P value of -2.91. Maximum ligand binding (65%) was observed in the presence of 50 mg of hydroxyapatite; a major portion of the activity was excreted through the kidneys. With the exception of excretory organs, gastrointestinal tract organs, including the liver, intestine, and colon, showed significant uptake; however, the bone uptake was low (<1%) at 30 min after the injection. The data were also confirmed by sequential imaging at 30-90 min following the intravenous injection. Conclusion: The high solubility and anionic properties of the complex led to major renal excretion and low hydroxyapatite uptake; therefore, the complex failed to demonstrate bone imaging behaviors.


Introduction
High morbidity and mortality rates of malignant diseases highlight the necessity of prompt diagnostic modalities, including positron emission tomography (PET). Limited access to radionuclide sources, such as cyclotrons and reactors, has led to the development of generatorbased PET radiopharmaceuticals.
In general, the high cost of installing and running cyclotrons in nuclear medicine centers is a source of major concern, especially in developing countries. Gallium-68 ( 68 Ga) generator is recognized as the most widely applicable PET generator. In fact, many generators have been developed by different companies, based on the organic and inorganic solid phases (1)(2)(3).
Bone metastases are common in the progression of various malignancies, such as prostate, breast, and lung carcinoma, often entailing progressive pain (4). Bisphosphonates such as alendronic acid (ALN) used in clinics have shown significant inhibitory binding constants (Ki) on bone resorption enzymes.
The idea of developing bone-avid agents for bone metastasis imaging has been of great interest to researchers. 68 Ga-based bone radiopharmaceuticals, including 68 Ga-EDTMP (5,6) and 68 Ga-BPAMD (7), have demonstrated remarkable preclinical and clinical performance. On the other hand, various ongoing research studies are focusing on more efficient radiotracers with rapid bone uptake, rapid washout, and reduced unnecessary soft tissue uptake.
Accordingly, another interesting approach is to develop bisphosphonate ligands, including metal chelating agents, such as diethylenetriaminepentaacetic acid (DTPA) moiety, as previously reported for the 99m Tc-labeled analog (8). However, many DTPA-based ALN ligands show no bone uptake with rapid renal excretion, possibly due to the anionic properties of DTPA moiety and its high water solubility (9,10).
According to previous research, a polycyclic dentate, containing a bisphosphonate ligand (DOTA), could be considered as a possibly suitable chelate to develop new bone-avid agents. This chelate forms stable chelates with various metals, including lanthanides, copper, and gallium and is developed by the conjugation of DOTA-NHS and [4-amino-1-hydroxy-1-(hydroxyoxido-phosphoryl)-butyl] phosphonic acid (ALN) (Figure 1).
In this study, we aimed to report the synthesis, characterization, and radiochemistry of [ 68 Ga]-DOTA-ALN ( Figure 1). For this purpose, biological evaluations and imaging studies were performed on normal rats.
Biodistribution data were acquired by counting normal saline-washed tissues after weighing on a Canberra TM high-purity germanium (HPGe) detector (model GC1020-7500SL); radionuclidic purity was assessed with the same detector. For activity measurement of the samples, a Capintec CRC radiometer (NJ, USA) was used.
Animal studies were performed in accordance with the United Kingdom Biological Council's Guidelines on the Use of Living Animals in Scientific Investigations (second edition). Images were acquired in the coincidence mode of a dual-head single-photon emission computed tomography (SPECT) system (DST-XL, Sopha Medical Vision, France).

Synthesis of DOTA-ALN
DOTA-ALN was synthesized by the conjugation of DOTA-NHS ligand to ALN base at equimolar levels. Briefly, ALN in form of monosodium triphosphate (1 mmol) was dissolved in 0.5 ml of 0.1 M HCl for better solubility at 25°C, followed by the addition of a few drops of trimethylamine. Then, DOTA-NHS (1 mmol) was dissolved in 0.5 ml of deionized water and stirred at 35-40°C for 20 min. The ALN solution was added dropwise to the latter mixture over 20 min, and the final mixture was stirred at 35°C for 24 h.
The excess amount of ALN was extracted by ethanol. For more purification, the yellow solid was washed several times with ether, methanol, and ethanol (yield: 80%; m.p.

Ge/ 68 Ga generator and quality control
The detailed quality control and application of the generator have been recently reported (6). A prototype 68 Ge/ 68 Ga SnO 2 -based generator (30 mCi) was purchased from Pars Isotope Co. (Tehran, Iran). For radiolabeling, the generator was eluted with 0.6 M of HCl. The eluate was analyzed to control radionuclide purity, using gamma spectroscopy with an HPGe detector, coupled with a Canberra TM multi-channel analyzer for 1000 sec. The breakthrough was measured by counting the same sample for 48 h after determining the small amount of 68 Ge in the sample. Chemical purity control was carried out to determine the amounts of Sn, Zn, Fe, Ge, and Ga ions, using the inductively coupled plasma atomic emission spectroscopy (ICP-OES) method. Radiochemical purity was assessed by ITLC chromatograms of 68 GaCl 3 solution in 10% ammonium acetate:methanol mixture on silica gel sheets. Also, 10 mM of DTPA solution (pH~4) was used on a Whatman No. 2 paper to check the percentage of colloidal 68 Ga fraction and/or noncationic gallium species.

Preparation of 68 Ga-DOTA-ALN
In a typical run, the acidic solution, containing 0.3 ml of [ 68 Ga]GaCl 3 (197.95 MBq, 5.35 mCi), was transferred to a 5 ml borosilicate vial, containing 150 mg of solid HEPES, and stirred at room temperature for 1 min. Afterwards, 100 µl of DOTA-ALN stock solution in deionized water (4 mg/ml ≈ 0.63 μmol) was added to the galliumcontaining vial and vortexed at 90 o C.
The active solution was assessed in terms of radiochemical purity by RTLC method at 5, 10, 20, and 30 min, respectively after labeling. The reaction mixture was then injected into a Sep-Pak C18 cartridge (Waters, USA), preconditioned with ethanol and water (2:5 ml), according to the manufacturer's protocol. The column was then flushed with air (10 ml), and the amount of trapped and passed activity was measured. The column was finally eluted with 0.5 ml fractions of ethanol:normal saline (1:9) solution, and the fraction with maximum activity was passed through a 0.22 μ antimicrobial filter into a sterilized vial.
The pH of the active solution was 5.5-6. A 5 μl sample of the final solution was spotted on the silica gel paper, and 10% ammonium acetate:methanol (1:1) mixture was used as the mobile phase. The ascent of the desired compound on the paper was compared with RTLC of [ 68 Ga]GaCl 3 and [ 68 Ga]-DOTA.

Calculation of partition coefficient
The partition coefficient (logP) of 68 Ga-DOTA-ALN was calculated, followed by the measurement of the specific activity ratio of organic and aqueous phases (P). A mixture of 1-octanol (1 ml) and isotonic acetate-buffered saline (1 ml, pH: 7), containing approximately 3.7 MBq of the radiolabeled gallium complex, was vortexed for 1 min at 37°C. Following centrifugation at > 1200 g for 5 min, octanol and aqueous phases were sampled and counted in an automatic well-type counter.

In vitro stability of 68 Ga-DOTA-ALN in the presence of human serum
The final solution (200 μCi, 50 μl) was incubated in the presence of freshly prepared human serum (300 μl) (Iranian Blood Transfusion Organization, Tehran, Iran) and maintained at 37°C for 2 hours. Every 30 min, 100 μl of 10% trichloroacetic acid was added to a portion of the mixture (50 μl), and the final mixture was centrifuged at 3000 rpm for 5 min, followed by decanting the supernatant from the debris. Stability was determined by performing frequent ITLC analysis of the supernatant, using the abovementioned ITLC system.

Hydroxyapatite (HA) binding assay
The HA binding assay was performed according to the procedure described in the literature (11), with slight modifications. In brief, 2 ml of saline solution (pH: 7.4) was added to vials, containing 1.0, 2.0, 5.0, 10.0, 20.0, and 50.0 mg of solid HA, respectively, and the mixture was shaken for 1 h. Then, 50 microliter of the radioactive preparation was added, and the mixture was shaken for 1 h at room temperature.
The suspension was centrifuged, and two aliquots of the supernatant liquid were obtained from each vial; radioactivity was measured with a well-type counter. Experiments were performed using a similar procedure in the absence of HA. The binding percentage to HA was calculated, using the following formula: where A is the mean radioactivity of the supernatant sample under study, and B denotes the mean total value of whole activity.

Biodistribution in wild-type rats
Distribution of the radiolabeled complex, as well as free 68 Ga cations, was determined in rat tissues. The total amount of radioactivity injected in each rat was measured by counting the 1 ml syringe before and after injection in a dose calibrator with fixed geometry. The animals were sacrificed by CO2 asphyxiation at specific time intervals after injection (n=5 for each time interval).
The tissues (blood, heart, lung, brain, intestine, faeces, skin, stomach, kidneys, liver, muscle, and bone tissues) were weighed and rinsed with normal saline. Their specific activities were determined with a HPGe detector, equipped with Asia Oceania J Nucl Med Biol. 2016; 4 (2) a sample holder. The values were expressed as the percentage of the injected dose per gram of tissue.

Imaging of 68 Ga-DOTA-ALN in wild-type rats
The volume of the final 68 Ga-DOTA-ALN solution (0.1 ml; 4.4 MBq activity) was injected into the dorsal tail vein of healthy rats. The total amount of the radioactive material, injected in each rat, was measured by counting the 1 ml syringe before and after injection, using an activity meter with fixed geometry.
The animals were relaxed by halothane and fixed in a suitable probe. Images were taken at 30, 60, and 90 min following the administration of the radiopharmaceutical in the coincidence mode. The useful field of view (UFOV) was 540×400 mm. The spatial resolution in the coincidence mode was 10 mm FWHM at CFOV, and sensitivity was 20 Kcps/µCi/cc. In total, 64 projections were acquired (30 s per view) with a 64×64 matrix.

Production of the precursor
Various reaction conditions were applied for DOTA-ALN conjugation. Conjugation was performed in dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and aqueous mixtures. The most appropriate reaction solvent was shown to be water, since reaction work-up seemed feasible without precipitation/purification processes or any concern about residual solvents (Figure 2). For better yields, the ALN-free base was added to DOTA-NHS (not vice versa).

Quality control of 68 Ga elution
For the quality control of 68 GaCl 3 solution, a timeactivity study was performed on the eluted sample after more than 10 half-lives of 68 Ga in order to check 68 Ge breakthrough. The data were recorded for eight days after elution. Calculations showed that the 68 Ge/ 68 Ga activity ratio was 1.6600×10 -5 at the time of elution.
The concentrations of tin (from the generator material), iron (from the sealing parts and acid impurities), zinc (as a decay product), and gallium (as the target material) were determined, using the ICP-OES method, except for iron with a concentration of nearly 0.23 mg/L; the concentration of the rest of ions was lower than 0.1 mg/L. The radiochemical purity of 68 GaCl 3 solution was assessed in two solvents, as shown in Table 1.

Radiolabeling
A series of reactions were performed in order  Asia Oceania J Nucl Med Biol. 2016; 4 (2) to reach the minimum required amount of the ligand; consequently, 0.5-0.6 µM of the ligand was applied for a typical labeling. Figure 3 demonstrates the chromatograms of 68 Ga-DOTA-ALN complex, as well as free 68 Ga under optimized conditions.

Partition coefficient
As expected from chemical behaviors, the lipophilicity of 68 Ga-DOTA-ALN was low due to the presence of three carboxylic groups in the DOTA moiety. The measured octanol-water partition coefficient (P) for the complex was found to depend on the pH of the solution. Based on the findings, at a pH of 7, the logP for the complex was -2.91.

Stability
The chemical stability of 68 Ga-DOTA-ALN was high enough to perform further analyses. Incubation of the complex in freshly prepared human serum (for 2 h at 37°C) showed no loss of 68 Ga from the complex. The radiochemical purity of the complex remained at the initial level for 2 h under physiological conditions ( Figure 4).

HA assay measurements
The HA assay demonstrated the low binding capacity of 68 Ga-DOTA-ALN complex to HA. Even with 50 mg of HA, less than 70% binding   therefore, bone-avidity behaviors were not observed in vitro ( Figure 5).

Biodistribution
Biodistribution studies were performed on 68 Ga-DOTA-ALN and free Ga 3+ (Figure 6). The radiolabeled complex was rapidly washed from blood circulation into the urinary tract with a very low uptake in the liver (Figure 7). For a better understanding of the uptake of other organs (except for kidneys), Figure  8 is presented. 68 Ga-DOTA-ALN imaging in wild-type rats is presented in Figure 9. The results were comparable with tissue dissection studies.

Discussion
The radiochemical purity of 68 GaCl 3 solution was assessed in two solvents. In 10 mmol/L of DTPA aqueous solution (solvent 1), free 68 Ga 3+ was coordinated to a more lipophilic moiety, as 68 Ga(DTPA) 2migrated to a higher Rf. The small radioactive fraction remaining at the origin could be attributed to colloids, since in the presence of very strong complexing agents (i.e., DTPA), ionic species other than 68 Ga(DTPA) 2can be rarely found.
Moreover, 10% ammonium acetate:methanol (1:1) mixture (solvent 2) was used for determining the radiochemical purity. The fast eluting species was possibly 68 Ga, and other ionic forms of 68 Ga, such as 68 GaCl 4 -(if existing), as well as colloids (not detected), remained at the origin (Rf= 0). The differences in impurity peaks in the two chromatograms could be partially related to colloidal impurity, which was insignificant. Also, the insignificant amount of activity (about < 1%) could be attributed to other ionic impurities.
Many considerations should be taken into account for radiolabeling. Some factors are known to influence the radiochemical purity of DOTA complex, while some change the quality of formulation for future applications (12). The elution portfolio of the generator is important, since more activity/eluted volume result in samples with higher specific activities for radiolabeling. On the other hand, use of 1 M or higher concentrations of HCl solutions usually yields a better radioactivity peak/elution, although other gallium species, which are not involved in radiolabeling, are formed at these concentrations.
In the presents study, by the selection of a suitable concentration (0.6-0.7 M), the major part of the daily-eluted generator activity was assessed; the peak of maximum activity ranged between 0.5 and 1.5 ml of the first elution. In the literature, the pH of radiolabeling for most ligands has been shown to be within the range of 4-5. Consequently, Ga activity eluted with 0.6-0.7 M of HCl usually exhibits a low pH (range: 1-2), which does not allow for the direct use of elution in the radiolabeling process. Therefore, addition of the calculated amount of solid HEPES to the elution has been proposed as a fast and reliable method.
The presence of metal impurities affects the complexation reaction due to impurities at ppm levels, compared to Ga-68 cations at ppb levels (or even lower levels). The radiolabeling yield usually decreases with the competition of other cations, particularly ferric cation in the radiolabeling reaction, leading to not only lower specific activities, but also a competition at the receptor level. Accordingly, the amount of cation release from the generator should be checked not only in the beginning of the generator application, but also in a routine manner in every center.
Long-term radiolysis reaction in the matrix of the generator is a major problem. Also, the interaction of Zn and Ga with DOTA moiety is reported to lead to an unwanted reaction with the conjugated peptide. In fact, routine (daily or twice a week) elution of the generator, even if not used for radiolabeling, would help to remove the unwanted cold cations present in the radiolabeling eluent.
In biodistribution studies, 68 Ga is majorly excreted from the gastrointestinal tract with high blood contents due to transferrin binding at early time intervals. In the present study, significant colon, bone, and stomach activities  (2) were observed, except in kidneys which are not significant accumulation sites ( Figure 6). The radiolabeled complex was rapidly washed from blood circulation into the urinary tract with a very low uptake in the liver (Figure 7). For a better understanding of the uptake of other organs except kidneys, the tracer biodistribution in other organs is presented in Figure 8 (after excluding the kidney uptake).
Based on the findings, less than 1% of the activity was accumulated in the bones. 68 Ga-DOTA-ALN imaging in wild-type rats showed highly comparable results with tissue dissection studies at 0.5 h following the injection, and the findings indicated high kidney uptake. However, at 1 h following the injection, kidney and bladder uptake was observed, although most of the activity was at the baseline level within 120 min (mostly detected in the bladder only) (Figure 8). 68 Ga-DOTA-ALN imaging in wild-type rats is presented in Figure 9. The results were comparable with tissue dissection studies. At 0.5 h after the injection, high kidney uptake was observed, while after 1 h following the injection, kidney and bladder uptake was reported, although most of the activity was at a baseline level after 120 min (mostly detected in the bladder only).

Conclusion
DOTA-ALN was completely synthesized and characterized in the present study. 68 Ga-DOTA-ALN was prepared within 7-10 min at 95°C with more than 99% radiochemical purity and 310-320 GBq/mmol specific activity. Also, water solubility properties and acceptable stability after solid phase purification were reported. The complex was stable up to 90 min with a logP of -2.91. Maximum ligand binding (65%) was observed in the presence of 50 mg of HA; a major portion of the activity was excreted through the kidneys. By excluding the excretory organs, gastrointestinal tract organs, including the liver, intestine, and colon, showed significant uptake; however; the bone uptake was low after 30 min (<1%). The data were confirmed by sequential imaging at 30-90 min following the injection. The high solubility and anionic properties of the complex led to high renal excretion and low HA uptake; therefore, the complex failed to demonstrate bone imaging behaviors.